Transcriptional Profiling and Consensus Molecular Subtype Assignment to Understand Response and Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Colorectal Cancer.

PURPOSE: Activating mutations in KRAS, NRAS, and BRAF are known to cause resistance to anti-epidermal growth factor receptor (EGFR) therapy; however, only approximately 40% of patients with colorectal cancer (CRC) with RASWT tumors respond to anti-EGFR treatment. We sought to discover novel biomarkers to predict response to anti-EGFR antibody treatment in CRC and to understand mechanisms of resistance to anti-EGFR therapy. MATERIALS AND METHODS: Transcriptomic profiles from three clinical and two preclinical cohorts treated with cetuximab were used to assign consensus molecular subtypes (CMS) to each sample and correlated with outcomes. RESULTS: Restricting to RASWT patients, we observed that CMS2 tumors (canonical subtype) had significantly higher response rates relative to other CMS when treated with cetuximab combination with doublet chemotherapy (Okita et al cohort: 92% disease control rate (DCR) for CMS2, chi-square P = .04; CALGB/SWOG 80405 cohort: 90% objective response rate (ORR) for CMS2, chi-square P < .001) and with single-agent cetuximab (68%, chi-square P = .01). CMS2 tumors showed best response among right-sided (ORR = 80%) and left-sided (ORR = 92%) tumors in the CALGB/SWOG 80405 cohort. CMS2 cells lines were most likely to be sensitive to cetuximab (60%) and CMS2 patient-derived xenograft had the highest DCR (84%). We found Myc, E2F, and mammalian target of rapamycin pathways were consistently upregulated in resistant samples (enrichment score >1, false discovery rate <0.25). Inhibitors of these pathways in resistant cell lines exhibited additive effects with cetuximab. CONCLUSION: These data suggest that CRC transcriptional profiles, when used to assign CMS, provide additional ability to predict response to anti-EGFR therapy relative to using tumor sidedness alone. Notably both right-sided and left-sided CMS2 tumors had excellent response, suggesting that anti-EGFR therapy be included as a treatment option for right-sided CMS2 tumors.

Oncogene addiction to GNAS in GNASR201 mutant tumors.

The GNASR201 gain-of-function mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins, driving oncogenesis in various low-grade/benign gastrointestinal and pancreatic tumors. In this study, we investigated the role of GNAS and its product Gαs in tumor progression using peritoneal models of colorectal cancer (CRC). GNAS was knocked out in multiple CRC cell lines harboring GNASR201C/H mutations (KM12, SNU175, SKCO1), leading to decreased cell-growth in 2D and 3D organoid models. Nude mice were peritoneally injected with GNAS-knockout KM12 cells, leading to a decrease in tumor growth and drastically improved survival at 7 weeks. Supporting these findings, GNAS overexpression in LS174T cells led to increased cell-growth in 2D and 3D organoid models, and increased tumor growth in PDX mouse models. GNAS knockout decreased levels of cyclic AMP in KM12 cells, and molecular profiling identified phosphorylation of ß-catenin and activation of its targets as critical downstream effects of mutant GNAS signaling. Supporting these findings, chemical inhibition of both PKA and ß-catenin reduced growth of GNAS mutant organoids. Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/ß-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors.

The anticancer plant triterpenoid, avicin D, regulates glucocorticoid receptor signaling: implications for cellular metabolism.

Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from "ancient hopanoids," avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use.

Natural triterpenoid avicins selectively induce tumor cell death.

Avicins, a family of plant-derived triterpenoids, have been shown to possess pro-apoptotic, anti-mutagenic and anti-inflammatory properties in mammalian cells. Through thiol binding, avicins can also mediate antioxidant defense. Accumulating evidence uncovered during the past several years suggests that avicins induce tumor cell death via multiple mechanisms. This review will focus on recent studies that provide insights into the cellular and molecular processes and pathways by which avicins induce tumor cell death, including the canonical intrinsic mitochondrial and the Fas-mediated apoptosis cascades as well as autophagy-associated non-apoptotic programmed cell death.

Red blood cell permeabilization by hypotonic treatments, saponin, and anticancer avicins.

Plasma membrane permeabilization by saponin and anticancer avicins was studied using light dispersion measurements, since high correlation between light dispersion changes and hemolysis has been demonstrated. Nevertheless, we observed that rat red blood cell swelling in moderately hypotonic media was accompanied by up to 20% decrease of light dispersion, when hemolysis was not yet detectable. Avicin G and avicin D were significantly more efficient than saponin in inducing cytotoxicity in PC3 human prostate cancer cells. We found that the preincubation of avicins with the plasma membrane, but not with the cytosolic fraction of previously lysed red blood cells, completely protected fresh cells against permeabilization. The data suggest that the plasma membrane can tightly bind the avicins, but not the saponin. Using the "osmotic protection" method with 100mOsm PEGs of increasing molecular weight in isotonic media, the size of the pores generated by avicin G and avicin D in the plasma membrane was estimated to be higher than the hydrodynamic radius of PEG-8000. The obtained results indicate that the anticancer activity of avicin G and avicin D could be related, at least partially, to their high ability to permeabilize biological membranes. These data might represent interest for possible applications of these anticancer drugs in vivo.

Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a) the cross-talk that occurs between redox and phosphorylation processes, and (b) the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

Avicin D, a plant triterpenoid, induces cell apoptosis by recruitment of Fas and downstream signaling molecules into lipid rafts.

Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-beta-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.

Avicin D selectively induces apoptosis and downregulates p-STAT-3, bcl-2, and survivin in cutaneous T-cell lymphoma cells.

Avicin D, a natural triterpenoid saponin, inhibits cell growth and induces apoptosis in transformed tumor cell lines in vitro and mouse skin carcinogenesis models in vivo. To investigate the anti-tumor effects of avicin D in cutaneous T-cell lymphomas (CTCL), we compared three CTCL cell lines and Sézary cells from three Sézary syndrome (SS) patients with normal CD4+ and activated CD4+ T cells from three healthy donors. Avicin D at 0.5-5 mug ml(-1) induced apoptosis in a time- and dose-dependent manner in three cell lines: MJ (-0.2 to 13% and 0.6-37%), Hut78 (2-39% and 3-53%), and HH (13-83% and 44-89%) at 24 and 48 hours, respectively. Avicin D at 0.5-5 microg ml(-1) for 48 hours caused more apoptosis in patients' Sézary cells than in healthy donors' CD4+ T cells and activated CD4+ T cells. The general caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Z-DEVD-FMK decreased avicin D-induced apoptosis in CTCL cells. Caspase-3 was activated and poly (ADP-ribose) polymerase was cleaved after avicin D treatment. Avicin D did not change the expression of signal transducer and activator of transcription-3 (STAT-3) but decreased phospho-signal transducer and activator of transcription-3 (p-STAT-3) protein levels in all three cell lines and two patients' Sézary cells. Avicin D also decreased expression of the inhibitor of apoptosis protein survivin, the anti-apoptotic protein bcl-2, but not the pro-apoptotic protein bax in these CTCL cells. In summary, avicin D selectively induced apoptosis, inhibited STAT-3 activation, and decreased apoptosis inhibitors (bcl-2 and survivin) in CTCL cell lines and SS patients' Sézary cells. Our findings underlie the therapeutic potential of avicin D in patients with SS.

Avicins, a novel plant-derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane.

Avicins are pro-apoptotic, anti-inflammatory molecules with antioxidant effects both in vitro and in vivo. Based on their ability to perturb mitochondrial functions and initiate apoptosis in tumor cells, we chose to study the bioenergetic effects of avicins on tumor cell mitochondria. Avicin-treated Jurkat cells, showed a decrease in the levels of cellular ATP as well as the rate of oxygen consumption. These effects on cellular metabolism appear to be a result of avicin's actions on the outer mitochondrial membrane (OMM). We speculate that avicins might initially inhibit the exchange of metabolites across the OMM leading to its subsequent permeabilization to cytochrome c. This speculation is supported by biophysical studies using lipid bilayers, which suggest that upstream of these effects, avicins target and close the voltage dependent anion channel (VDAC). Closure of VDAC would lead to an overall lowering of the cell energy metabolism, subsequently pushing these cells towards the apoptotic pathway by permeabilization of the OMM and release of cyt-c. Avicins therefore not only represent a novel pharmacological tool for treatment of cancers, but also highlight the influence ancient plant metabolites could have on human health.

Avicins, natural anticancer saponins, permeabilize mitochondrial membranes.

Avicins are a class of natural saponins with selective pro-apoptotic activity in cancer cells. In this work, we studied the influence of avicins on metabolic state of rat liver mitochondria. Avicin-treated mitochondria underwent a significant decrease in oxygen consumption rate that was completely restored by addition of exogenous cytochrome c. On the other hand, avicins increased the rotenone-insensitive oxidation of external NADH in the presence of exogenous cytochrome c, long before high amplitude swelling of mitochondria was observed. The increase in external NADH oxidation was cyclosporin A-insensitive. Avicin G significantly accelerated hydroperoxide-induced oxidation of mitochondrial endogenous NAD(P)H, the drop of the inner membrane potential and the high amplitude swelling of mitochondria. The obtained data might explain selective induction of apoptosis in tumor cells by avicins. Based on other studies showing that tumor cells generate hydroperoxides with a very high rate, avicins could provide a new strategy of anticancer therapy by sensitizing cells with high levels of reactive oxygen species to apoptosis.

Effects of the tumor inhibitory triterpenoid avicin G on cell integrity, cytokinesis, and protein ubiquitination in fission yeast.

Avicins comprise a class of triterpenoid compounds that exhibit tumor inhibitory activity. Here we show that avicin G is inhibitory to growth of the fission yeast Schizosaccharomyces pombe. S. pombe cells treated with a lethal concentration of avicin G (20 microM) exhibited a shrunken morphology, indicating that avicin G adversely affects cell integrity. Cells treated with a sublethal concentration of avicin G (6.5 microM) exhibited a strong cytokinesis-defective phenotype (multiseptated cells), as well as cell morphology defects. These phenotypes bear resemblance to those resulting from loss of Rho1 GTPase function in S. pombe. Indeed, Rho1-deficient S. pombe cells were strongly hypersensitive to avicin G, suggesting that the compound may perturb Rho1-dependent processes. Consistent with previously observed effects in human Jurkat T cells, avicin G treatment resulted in hyperaccumulation of ubiquitinated proteins in S. pombe cells. Interestingly, proteasome-defective S. pombe mutants were not markedly hypersensitive to avicin G, whereas an anaphase-promoting complex (mitotic ubiquitin ligase) mutant exhibited avicin G resistance, suggesting that the increase in levels of ubiquitinated proteins resulting from avicin G treatment may be due to increased protein ubiquitination, rather than inhibition of 26S proteasome activity. Mutants defective in the cAMP/PKA pathway also exhibited resistance to avicin G. Our results suggest that S. pombe will be a useful model organism for elucidating molecular targets of avicin G and serve as a guide to clinical application where dysfunctional aspects of Rho and/or ubiquitination function have been demonstrated as in cancer, fibrosis, and inflammation.

Avicinylation (thioesterification): a protein modification that can regulate the response to oxidative and nitrosative stress.

Avicins are a recently discovered family of plant-derived terpenoid molecules that possess proapoptotic, antiinflammatory, and cytoprotective properties in mammalian cells. Previous work demonstrating that avicins can exert their effects by suppressing or activating the redox-sensitive transcription factors NF-kappaB and nuclear factor-erythroid 2 p45-related factor (Nrf2), respectively, has raised the idea that they may react with critical cysteine residues. To understand the molecular mechanism through which avicins regulate protein function, we examined their effects on the paradigmatic redox-responsive transcriptional activator, OxyR of Escherichia coli, which protects bacterial cells against oxidative and nitrosative stresses. In vitro transcription assays demonstrated that avicins activate OxyR and its target genes katG and oxyS in a DTT-reversible manner. In addition, katG-dependent hydroperoxidase I activity was enhanced in avicin-treated bacteria. Mass spectrometric analysis of activated OxyR revealed thioesterification of the critical regulatory cysteine, Cys-199, to an avicin fragment comprising the outer monoterpene side chain. Our results indicate that avicinylation can induce adaptive responses that protect cells against oxidative or nitrosative stress. More generally, transesterification may represent a previously undescribed thiol-directed posttranslational modification, which extends the code for redox regulation of protein function.

Mitochondria as integrators of information in an early-evolving animal: insights from a triterpenoid metabolite.

Mitochondria have the capacity to integrate environmental signals and, in animals with active stem cell populations, trigger responses in terms of growth and growth form. Colonial hydroids, which consists of feeding polyps connected by tube-like stolons, were treated with avicis, triterpenoid electrophiles whose anti-cancer properties in human cells are mediated in part by mitochondria. In treated hydroids, both oxygen uptake and mitochondrial reactive oxygen species were diminished relative to controls, similar to that observed in human cells exposed to avicins. While untreated colonies exhibit more stolon branches and connections in the centre of the colony than at the periphery, treated colonies exhibit the opposite: fewer stolon branches in the centre of the colony than at the periphery. The resulting growth form suggest an inversion of the normal pattern of colony development mediated by mitochondrial and redox-related perturbations. An as-yet-uncharacterized gradient within the colony may determine the ultimate phenotypic effects of avicin perturbation.

Triterpenoid electrophiles (avicins) suppress heat shock protein-70 and x-linked inhibitor of apoptosis proteins in malignant cells by activation of ubiquitin machinery: implications for proapoptotic activity.

Avicins are plant-derived triterpenoid stress metabolites that have both proapoptotic and cytoprotective properties. Avicins induce apoptosis in Jurkat T leukemia cells by targeting mitochondria and release of cytochrome c that occurs in a p53-independent manner. However, postmitochondrial antiapoptotic barriers, such as increased expression of heat shock proteins (Hsp) and X-linked inhibitor of apoptosis proteins (XIAP), frequently exist in cancer cells and often account for resistance to chemotherapy and a poor prognosis. In this article, we show the role of avicins in the activation of stress-regulated ubiquitination and degradation of Hsp70 and XIAP. This is the first report showing the regulation of Hsp70 via the ubiquitin/proteasome pathway. We also show the induction of E3alpha ubiquitin ligase in avicin-treated Jurkat T leukemia cells, and its involvement in the degradation of XIAP. Avicin-mediated suppression of Hsp70 and XIAP was further confirmed in other leukemic/lymphoma cell lines and freshly isolated peripheral blood lymphocytes from Sezary syndrome patients. No change in the Hsp70 and XIAP proteins was observed in peripheral blood lymphocytes from normal donors. We propose that the ability of avicins to induce ubiquitination and regulate the degradation of Hsp70 and XIAP in leukemia cells could have important implications in the treatment of drug-resistant neoplasia and inflammatory disorders.

Proapoptotic triterpene electrophiles (avicins) form channels in membranes: cholesterol dependence.

Avicins, a family of triterpenoid saponins from Acacia victoriae, can regulate the innate stress response in human cells. Their ability to induce apoptosis in transformed cells makes them potential anticancer agents. We report that avicins can form channels in membranes. The conductance reached a steady state after each addition, indicating a dynamic equilibrium between avicin in solution and in the membrane. The high power dependence (up to 10) of the membrane conductance on the avicin concentration indicates the formation of multimeric channels, consistent with the estimated pore radius of 1.1 nm. This radius is too small to allow protein flux across the mitochondrial outer membrane, a process known to initiate apoptosis. Channel formation is lost when avicin's amphipathic side chain is removed, implicating this as the channel-forming region. A small difference in this side chain results in strong cholesterol dependence of channel formation in avicin G that is not found in avicin D. In neutral membranes, avicin channels are nonselective, but negatively-charged lipids confer cation selectivity (5:1, K(+):Cl(-)), indicating that phospholipids form part of the permeation pathway. Avicin channels in the mitochondrial outer membrane may favor apoptosis by altering the potential across this membrane and the intermembrane space pH.

Triterpenoid electrophiles (avicins) activate the innate stress response by redox regulation of a gene battery.

Avicins are proapoptotic and anti-inflammatory triterpene electrophiles isolated from an Australian desert tree, Acacia victoriae. The presence of two alpha,beta unsaturated carbonyl groups (Michael reaction sites) in the side chain of the avicin molecule prompted us to study its effects on NF-E2-related factor 2 (Nrf2), a redox-regulated transcription factor that controls the expression of a battery of detoxification and antioxidant proteins via its binding to antioxidant response element (ARE). Avicin D-treated Hep G2 cells showed translocation of Nrf2 into the nucleus and a time-dependent increase in ARE activity. These properties were sensitive to DTT, suggesting that avicins affect one or more critical cysteine residues, probably on the Keap1 molecule. Downstream of ARE, an activation of a battery of stress-induced proteins occurred. The implications of these findings were evaluated in vivo in mouse skin exposed to an ancient stressor, UV light. Avicins inhibited epidermal hyperplasia, reduced p53 mutation, enhanced apoptosis, decreased generation of 8-hydroxy-2'-deoxyguanosine, and enhanced expression of NADPH:quinone oxidoreductase 1 and heme oxygenase-1. These data, combined with our earlier published work, demonstrate that avicins represent a new class of plant stress metabolites capable of activating stress adaptation and suppressing proinflammatory components of the innate immune system in human cells by redox regulation. The relevance for treatment of clinical diseases in which stress responses are dysfunctional or deficient is discussed.

Isolation and structures of avicins D and G: in vitro tumor-inhibitory saponins derived from Acacia victoriae.

Two new saponins named avicins D (1) and G (2) were isolated from the seed pods of the desert legume plant Acacia victoriae. The structures, elucidated by 1D and 2D NMR studies and by chemical means, were characterized as acacic acid-bearing oligosaccharides at C-3 and C-28 with a side chain linked to C-21 comprised of two monoterpene carboxylic acids and a quinovose moiety. Both compounds exhibited potent cytotoxicity (apoptosis) against human T-cell leukemia (Jurkat cells) in vitro.

Metabolomics of plant saponins: bioprospecting triterpene glycoside diversity with respect to mammalian cell targets.

One of the goals of cancer chemotherapy and prevention is the discovery of compounds that are relatively selective to tumor cells and, therefore, have reduced effects on normal cell growth. In previously published studies, it was shown that certain triterpene saponins (called avicins) from a desert tree, Acacia victoriae, are selectively toxic to tumor cells at very low doses (IC(50): 0.2 microg/mL for Jurkat cells). To extend this research to human clinical studies, we needed to find a reliable supply of avicins and have developed a transformed "hairy root" culture as a means of biomass production. Protocols were optimized for A. victoriae micropropagation; after a boiling water treatment, A. victoriae seeds were maintained under in vitro conditions on defined media. Embryo-axis explants from shoot tips were removed and infected with Agrobacterium rhizogenes Conn (R 1000) for hairy root induction. Plasmid integration was confirmed by PCR analysis with a primer set for a segment of the rol B gene. Culture conditions have been optimized for root biomass production, and various inducers have been investigated for enhancement of avicin production. Hairy root cultures were compared with intact pod tissue from field-grown sources for avicin content following partial purification of triterpene glycosides and HPLC separation of the secondary metabolites. From bioassays of the collected HPLC fractions, we have identified putative triterpene "metabolic clusters" with enhanced activity against tumor cells. This now provides a system for both production of clinical trial lots of active samples, but also a means to correlate structure of individual triterpene glycosides with specific cellular target activity in mammalian cells.